Tropical Biomedicine 36(4): 926–937 (2019)

A method for distinguishing the important vectors dirus and An. cracens (Diptera: Culicidae) based on antennal sensilla of adult females

Taai, K.1, Harbach, R.E.2, Somboon, P.3, Sriwichai, P.4, Aupalee, K.3, Srisuka, W.5, Yasanga, T.6, Phuackchantuck, R.7, Jatuwattana, W.3, Pusawang, K.3 and Saeung, A.3* 1Faculty of Veterinary Medicine, Western University, Kanchanaburi 71170, Thailand 2Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK 3Center of Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 4Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand 5Entomology Section, Queen Sirikit Botanic Garden, Chiang Mai 50180, Thailand 6Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 7Research Administration Sections, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand *Corresponding author e-mail: [email protected] Received 25 February 2019; received in revised form 26 June 2019; accepted 28 June 2019

Abstract. Some species of the Anopheles dirus species complex are considered to be highly competent malaria vectors in Southeast Asia. Anopheles dirus is the primary vector of Plasmodium falciparum and P. vivax while An. cracens is the main vector of P. knowlesi. However, these two species are difficult to distinguish and identify based on morphological characters. Hence, the aim of this study was to investigate the potential use of antennal sensilla to distinguish them. Large sensilla coeloconica borne on the antennae of adult females were counted under a compound light microscope and the different types of antennal sensilla were examined in a scanning electron microscope. The antennae of both species bear five types of sensilla: ampullacea, basiconica, chaetica, coeloconica and trichodea. Observations revealed that the mean numbers of large sensilla coeloconica on antennal flagellomeres 2, 3, 7, 10 and 12 on both antennae of both species were significantly different. This study is the first to describe the types of antennal sensilla and to discover the usefulness of the large coeloconic sensilla for distinguishing the two species. The discovery provides a simple, reliable and inexpensive method for distinguishing them.

INTRODUCTION to eliminate malaria by 2020. In Thailand, the total number of confirmed malaria cases in Malaria is a disease caused by plasmodial 2016 was 37,209 (Bureau of Vector Borne parasites that are transmitted to humans Diseases, Ministry of Public Health, 2017). through the bites of female Anopheles Seven taxonomic groups are known to mosquitoes. There were approximately 219 include the main malaria vectors in Southeast million malaria cases in 90 countries and an Asia, i.e. the Culicifacies, Dirus, Fluviatilis, estimated 435,000 deaths due to the disease Leucosphyrus, Minimus and Sundaicus in 2017 (WHO, 2018). For example, 1,398 Complexes, and the Maculatus Group cases of falciparum malaria were reported (Manguin et al., 2008). According to Sallum in 28 provinces of China in 2011, especially et al. (2005), the Dirus Complex comprises in epidemic areas of Yunnan and Hainan seven species: An. baimaii Sallum & provinces (WHO, 2018). China is now aiming Peyton, An. cracens Sallum & Peyton, An.

926 dirus Peyton & Harrison, An. elegans James, MATERIALS AND METHODS An. nemophilous Peyton & Ramalingam, An. scanloni Sallum & Peyton and An. Mosquitoes and species identification takasagoensis Morishita. Takano et al. (2010) Specimens of laboratory strains of An. dirus added an eighth species, informally denoted were originally collected in Mae Sod District, as “aff. takasagoensis”. Anopheles dirus has Tak Province, Thailand. Specimens of free- a wide distribution in eastern Asia, being mating An. cracens were originally from recorded in Cambodia, China (Yunnan the Armed Forces Research Institute of Province and Hainan Island), Laos, Myanmar, Medical Sciences laboratory, Bangkok, Thailand and Vietnam. Anopheles cracens Thailand. Anopheles dirus (Hainan strain, occurs southward from southern Thailand China) was obtained from the Department of through peninsular Malaysia (Perlis, Vector Ecology and Environment, Nagasaki Terengganu, Kuala Lipis Pahang states) into University, Japan. The species were identified Sumatra, Indonesia (Sallum et al., 2005; based on morphology (Rattanarithikul et al., Jiram et al., 2012). 2006; Somboon and Rattanarithikul, 2013) and Anopheles dirus and An. baimaii have the allele-specific polymerase chain reaction been incriminated as the principle vectors (AS-PCR) method of Walton et al. (1999). of the malarial protozoa Plasmodium Briefly, genomic DNA was extracted from falciparum and P. vivax in China and wings and legs of individual mosquitoes Southeast Asia (Manguin et al., 2008; using the PureLink® Genomic DNA Kit Saeung, 2012; Tainchum et al., 2015), while (Invitrogen, USA), according to the manu- An. cracens has been found to be the main facturer’s recommendations. Each PCR vector of P. knowlesi in peninsular Malaysia reaction was conducted using a 25 µl volume (Pahang) (Vythilingam et al., 2008; Jiram et containing 0.5 U of Taq DNA polymerase al., 2012). (Invitrogen, USA), 1x Taq buffer, 1.5 mM of Undoubtedly, correct identification of MgCl2, 0.2 mM of dNTP, 0.25 µM of each vector species is important for proper primer and 1 µl of the extracted DNA. The management and control of malaria. How- amplification profile consisted of an initial ever, identification of members of the Dirus denaturation at 94°C for 5 min, 35 cycles Complex is difficult due to overlapping at 94°C for 30 s, 53°C for 30 s, 72°C for 30 s morphological characters (Hii and Rueda, and a final extension at 72°C for 5 min. The 2013). Various methods for recognition and amplified products were electrophoresed confirmation of the taxonomic status of the on 1.5% agarose gel and stained with SYBR® individual species have been investigated, Safe DNA gel stain (Invitrogen, USA). including cytogenetics (Baimai, 1988; Baimai, 1998), enzyme electrophoresis rearing (Green et al., 1992) and molecular genetics A colony of each of the three strains was (Walton et al., 1999; Sallum et al., 2005; established and maintained in an insectary Phunngam et al., 2017). Furthermore, of the Department of Parasitology, Faculty Somboon et al. (2009) investigated the of Medicine, Chiang Mai University, using structure of the cibarial armature for the procedures described by Choochote and distinguishing four species of the Dirus Saeung (2013). The insectary was maintained Complex but no significant differences were at 27±2°C, 70–80% relative humidity and observed. However, the antennal sensilla illuminated by a combination of natural of members of the complex have not been daylight from a glass window and fluorescent investigated. In the present study, the various lighting provided for approximately 12 h a types of sensilla borne on the antennae of day. females of An. dirus and An. cracens were examined using scanning electron micro- Light microscopy scopy, and are described here for the first Large sensilla coeloconica (lco) on each time. antennal flagellum of five-day-old female

927 mosquitoes were observed using an Olympus sputter-coated with gold. Sensilla were BX53 compound microscope. Individual observed and photographed in a JEOL- specimens were immersed in 10% potassium JSM6610LV scanning electron microscope. hydroxide (KOH) solution in a small bottle and held in an oven at 45°C for 30–45 min. Statistical analysis After clearing, they were washed with 80% The number of large sensilla coeloconica on ethanol, their antennae were removed the antennae of specimens of An. cracens using an insect needle and the two antennae and the two strains of An. dirus were of each female were mounted together on analyzed using the Kruskal-Wallis test. A a microscope slide with Neo-shigaral post-hoc Dunn’s test was used for multiple medium (Tokyo, Japan). The large sensilla comparisons of means. All data were coeloconica borne on the left and right analyzed using IBM SPSS statistics, version flagellum of 30 females of each strain were 24 for Windows (SPSS Inc., Chicago). The counted (n = 60 flagella/strain) (Taai et al., level of significance was set at 5% (p-value 2017). < 0.05).

Scanning electron microscopy Thirty heads of four- to five-day-old females RESULTS of each strain were removed under a stereomicroscope and rinsed three times in Molecular identification phosphate buffer (pH 7.4) to remove surface The AS-PCR confirmed the morphological debris. The heads were then dehydrated identification of An. dirus (Thailand and through an ethanol series of 35, 70, 80 (10 Hainan strains) and An. cracens. PCR min, two changes) and 95% (15 min, two species-specific products were 562 bp and changes), followed by absolute ethanol 514 bp for An. dirus (both strains) and (10 min, two changes), and then dried in a An. cracens, respectively (Figure 1). critical point dryer. The antennae were carefully dissected from the head capsule Morphology of antennal sensilla under a stereomicroscope, as described by In general, the antennae of female mosquitoes Hempolchom et al. (2017). The antennae consist of two basal segments (scape and were mounted on aluminum stubs with pedicel) and an elongate segmented double-sided carbon adhesive tape and flagellum (Figures 2A–2C). The scape is the

Figure 1. Gel of allele-specific PCR for distinguishing An. dirus and An. cracens. Lanes 1 and 2: An. dirus, Thailand strain (562 bp); lanes 3 and 4: An. dirus, Hainan strain (562 bp); lanes 5 and 6: An. cracens, Thailand strain (514 bp); lane M: 100 bp ladder.

928 Figure 2. Scanning electron micrographs of the antenna of females of An. dirus (virtually identical in An. cracens). (A) The flagellum consisting of 13 flagellomeres. (B) The scape (Sc), pedicel (Pe) and first flagellomere (I), densely covered with aculeae (ac). (C) The basal plate (Bpl) of flagellomere I connected with the pedicel. basal collar-shaped segment (Figure 2B). The pedicel is a large globular segment (Figure 2B) that bears the flagellum (Figure 2C). Each flagellum consists of 13 flagellomeres (Figure 2A). Aculeae (ac, microtrichium-like spicules) densely cover the surface of the scape, pedicel and the first flagellomere (Figures 2B and 2C). Based on shape and size, five types of sensilla are borne on the antennae of An. dirus and An. cracens: sensilla ampullacea, sensilla basiconica, sensilla chaetica, sensilla coeloconica and sensilla trichodea (Figure 3). The morphology of each sensillum type is similar in both species. Sensilla ampullacea are small peg organs in deep pits with a narrow opening. This type of sensillum is abundant on the first antennal flagellomere and decreases in number on flagellomeres 2–5 (Figures 3 and 4A). Sensilla chaetica are long, thick-walled, Figure 3. Scanning electron micrograph sharp-pointed setae that arise from sockets. showing the various types of sensilla borne on There are two types: large and small (Figure the antennae of females of An. dirus and An. 3). Six large sensilla chaetica are borne in cracens. ac, aculeae; btc, blunt-tipped sensillum whorls proximally on flagellomeres 2–13. The trichodeum; lch, large sensillum chaeticum; small sensilla chaetica usually occur on the lco, large sensillum coeloconicum; ltc, long distal ends of flagellomeres 2–13. Both types sharp-tipped sensillum trichodeum; sa, sensillum ampullaceum; sb, sensillum basi- also occur on the ventral surface of the first conicum; sch, small sensillum chaeticum; stc, flagellomere and are often interspersed with short sharp-tipped sensillum trichodeum. aculeae (Figure 3).

929 Figure 4. Scanning electron micrographs of antennal sensilla of females of An. dirus and An. cracens. (A) Sensillum ampullaceum. (B) Small sensillum coeloconicum. (C) Short form of large sensillum coeloconicum, peg not reaching the orifice of the pit. (D) Long form of large sensillum coeloconicum, peg extending beyond the orifice of the pit. (E) Sensillum basiconicum (grooved peg).

Sensilla trichodea are the most abundant Sensilla coeloconica are thick-walled sensilla found on the flagellum of both sensilla. Two types, large and small, can be species. They arise from a small prominent distinguished based on shape. Large sensilla base and have a smooth surface. Three coeloconica are peg-shaped projections types of sensilla trichodea are present: long located in deep depressions. They have sharp-tipped trichodea, short sharp-tipped 10–14 deep longitudinal grooves on their trichodea and blunt-tipped trichodea. The surfaces (Figure 3). The pegs may or may long sharp-tipped trichodea often bend not project from the floor of the depression toward the apex (Figure 3) and their number through the circular openings at the surface increases from the proximal to the distal ends of the cuticle (Figures 4C and 4D). Both of flagellomeres in both species. The short straight and curved-tipped sensilla sharp-tipped trichodea of both species are coeloconica are found on the antennae of slightly bent and are fewer in number than both species. Small sensilla coeloconica the former type (Figure 3). Blunt-tipped arise from the bottom of a shallow pit, but trichodea are shorter in length, have rounded they do not protrude from the opening of tips, and have nearly the same diameter from the pit. These sensilla have a volcano-like the base to the tip (Figure 3). They also occur structure with a small opening at the tip and in fewer numbers than the sharp-tipped a much smaller cuticular opening at the trichodea in both species. surface than large coeloconica. This type of

930 Figure 5. Scanning electron micrographs showing the different numbers of large sensilla coeloconica (lco) observed on flagellomere 2 of antennae of females of (A) An. dirus and (B) An. cracens.

Figure 6. Scanning electron micrographs of flagellomere 13. (A, B) Absence of large sensilla coeloconica in Thai An. dirus and Hainan An. dirus, respectively. (C) Presence of large sensillum coeloconicum (circled) in An. cracens. (D) Higher magnification of sensilla coeloconica (sco) at the tip of flagellomere 13. sensillum coeloconicum occurs on the first at the apex, and are scattered on the flagellomere (Figures 3 and 4B), and the tip surfaces of all flagellomeres of both of flagellomere 13 (Figure 6D). species (Figures 3 and 4E). Sensilla basiconica are curved peg-like or horn-shaped sensilla. The surface of Number of large coeloconic sensilla on sensilla basiconica is grooved lengthwise antennae of females similar to those of sensilla coeloconica, but The number of large sensilla coeloconica per the grooves are not deep and are fewer in antennal flagellomere ranges from 0–6 in number (10–12). They arise from small An. dirus (both strains) and 0–5 in An. prominences within an ill-defined alveolus cracens. The largest number of large sensilla (Figures 3 and 4E). Sensilla basiconica are coeloconica occurs on flagellomere 2 of both slender, slightly bent, tapered and pointed strains of An. dirus (Table 1 and Figure 5).

931 Table 1. Mean numbers of sensilla coeloconica on antennal flagellomeres 1–13 of females of Thai An. dirus (DTH), Hainan An. dirus (DHN) and An. cracens (CR) (30 females/strain, n = 60)

Mosquito species Flagellomere Kruskal-Wallis Dunn’s test DTH DHN CR Test (range) (range) (range)

1 4.13±0.75 3.02±1.17 2.75±0.79 CR vs DHN (P = 0.288) p < 0.001* (2–6) (1–6) (1–5) CR vs DTH (P < 0.001) DHN vs DTH (P < 0.001)

2 4.03±0.86 3.92±1.00 2.95±0.72 CR vs DHN (P < 0.001) p < 0.001* (2–6) (2–6) (1–4) CR vs DTH (P < 0.001) DHN vs DTH (P=1.000)

3 2.80±0.84 2.98±1.05 1.83±0.69 CR vs DHN (P < 0.001) p < 0.001* (1–4) (1–5) (0–3) CR vs DTH (P < 0.001) DHN vs DTH (P = 1.000)

4 2.37±0.74 2.77±0.93 1.93±0.73 CR vs DHN (P < 0.001) p < 0.001* (1–4) (1–5) (0–3) CR vs DTH (P = 0.019) DHN vs DTH (P = 0.037)

5 1.47±0.62 1.93±0.84 1.28±0.56 CR vs DHN (P < 0.001) p < 0.001* (1–3) (1–4) (0–3) CR vs DTH (P = 0.395) DHN vs DTH (P = 0.003)

6 1.55±0.65 1.88±0.78 1.45±0.54 CR vs DHN (P = 0.007) p = 0.005* (1–3) (1–4) (0–3) CR vs DTH (P = 1.000) DHN vs DTH (P = 0.042)

7 1.32±0.47 1.13±0.60 0.73±0.63 CR vs DHN (P = 0.002) p < 0.001* (1–2) (0–3) (0–2) CR vs DTH (P < 0.001) DHN vs DTH (P = 0.236)

8 1.15±0.40 1.28±0.52 1.03±0.45 CR vs DHN (P = 0.010) p = 0.014* (0–2) (0–2) (0–2) CR vs DTH (P = 0.604) DHN vs DTH (P = 0.301)

9 1.00±0.18 0.78±0.45 0.72±0.49 CR vs DCH (P = 1.000) p < 0.001* (0–2) (0–2) (0–2) CR vs DTH (P = 0.000) DHN vs DTH (P = 0.011)

10 1.20±0.40 1.28±0.52 0.98±0.39 CR vs DHN (P = 0.001) p < 0.001* (1–2) (0–2) (0–2) CR vs DTH (P = 0.036) DHN vs DTH (P = 0.819)

11 1.82±0.43 1.53±0.62 1.15±0.58 CR vs DHN (P = 0.001) p < 0.001* (1–3) (0–3) (0–3) CR vs DTH (P < 0.001) DHN vs DTH (P = 0.029)

12 2.15±0.44 2.27±0.71 2.77±0.50 CR vs DHN (P < 0.001) p < 0.001* (1–3) (0–4) (2–4) CR vs DTH (P < 0.001) DHN vs DTH (P = 0.398)

13 0.35±0.58 CR vs DHN (P = 1.000) 00p < 0.001* (0–2) CR vs DTH (P < 0.001) DHN vs DTH (P < 0.001)

Total 25.33±2.70 24.78±3.54 19.58±2.57 CR vs DHN (P < 0.001) p < 0.001* (range) (19–31) (17–32) (12–26) CR vs DTH (P < 0.001) DHN vs DTH (P = 1.000)

932 The mean number of large sensilla Group in Thailand based on antennal sensilla coeloconica borne on each of flagellomeres of females. More recently, Taai et al. (2017) 2, 3, 7, 10 and 12 in An. dirus (both strains) reported an effective method based on and An. cracens is significantly different antennal sensilla for the identification and (Table 1). Likewise, the mean number of separation of females of An. minimus, the large sensilla coeloconica per flagellum of primary vector of malaria in Thailand, and An. dirus (Thailand strain, 25.33; Hainan its sister species An. harrisoni. From these strain, 24.78) and An. cracens (19.58) is studies, it would seem that antennal sensilla significantly different (Dunn’s test, p < 0.0001) are useful structures for distinguishing (Table 1). Interestingly, large sensilla malaria vectors of species complexes. coeloconica were found on flagellomere rely on olfactory information for 13 of Thai An. dirus, with a range of 0–2 locating hosts, mating and locating suitable (Figure 6C), but were always absent in oviposition sites (Hildebrand and Shepherd, Hainan An. dirus and An. cracens (Table 1, 1997; Qiu et al., 2006). Olfactory receptor Figures 6A and 6B). neurons in insects are contained in sensilla on antennae and mouthparts (Qiu et al., 2006). In the present study, we describe the ultra- DISCUSSION structure of five types of sensilla (ampullacea, basiconica, chaetica, coeloconica and Based on morphology, the two sibling trichodea) on the antennae of females of species, An. cracens and An. dirus, are two important malaria vectors, An. dirus difficult to distinguish unambiguously. (Thailand and Hainan strains) and An. Molecular methods are now widely used cracens. The morphology of these sensilla to distinguish and identify them (Walton et agrees with the findings of earlier studies al., 1999; Sallum et al., 2005; Sallum et al., on mosquitoes (McIver, 1982; Sutcliffe, 1994; 2007; Phunngam et al., 2017). In addition to Pitts and Zwiebel, 2006; Hill et al., 2009; molecular methods, Cui et al. (1992) used Hempolchom et al., 2017). Sensilla trichodea gas chromatography of cuticular hydro- are the most abundant sensilla on the carbons to distinguish members of the Dirus antennae of An. dirus and An. cracens, which Complex in Hainan Province of China, but no is also true of other insects, such as muscid significant differences were found between (Diptera: Muscidae) (Wang et al., 2014), them. However, these advanced methods, yellow dung flies (Diptera: Scathophagidae) need to be performed in the laboratory at (Liu et al., 2016) and other mosquitoes high cost. Thus, alternative morphological (Pitts and Zwiebel, 2006; Hill et al., 2009; structures (e.g. antennal sensilla, cibarial Seenivasagan et al., 2009; Schultze et al., armature, wings, etc.) have been success- 2014). Onagbola and Fadamiro (2008) fully used for distinguishing isomorphic or noted that sensilla trichodea are putative cryptic species (Somboon et al., 2001; Pitts mechanoreceptors. Schultze et al. (2014) and Zwiebel, 2006; Saeung et al., 2014; Wijit confirmed that the blunt-tipped sensilla et al., 2016; Wike et al., 2016; Sumruayphol et trichodea of female mosquitoes may respond al., 2016; Hempolchom et al., 2017; Taai et to oviposition site-related compounds. al., 2017). Saeung et al. (2014) constructed Sensilla ampullacea are few in number a robust key for the identification of eight on the flagellum of both An. dirus and An. species of the Hyrcanus Group (An. cracens, and this type of sensillum is similar argyropus, An. crawfordi, An. nigerrimus, in structure to those reported for other species An. nitidus, An. paraliae, An. peditaeniatus, of Anopheles (Boo and Mclver, 1975; Taai et An. pursati and An. sinensis) based on al., 2017). This type of sensillum has been morphometrics and ultrastructure of eggs classified as hygro- and thermoreceptors. observed using scanning electron micro- Like sensilla trichodea, sensilla chaetica scopy. Subsequently, Hempolchom et al. are putative mechanoreceptors (Hill et al., (2017) constructed a key to reliably 2009). Additionally, this sensillum type is distinguish eight species of the Hyrcanus presumed to function as a contact chemo-

933 receptor during oviposition (Seenivasagan et cracens. This study shows that both species al., 2009). The sensilla basiconica observed bear morphologically similar antennal on the antennae of An. dirus and An. cracens sensilla, with marked differences in the females correspond with type I grooved pegs number of large coeloconic sensilla on observed in eight species of the Hyrcanus each flagellomere and on both flagella. This Group (set on small prominences within an finding provides an alternative method for ill-defined alveolus) by Hempolchom et al. distinguishing the two sibling species. The (2017). Several authors have suggested that mean total number of large sensilla basiconica are olfactory sensilla (Mclver, coeloconica on antennae of An. cracens is 1974), which respond to vapors of ammonia, less than in An. dirus. This method is simple, acetone and water (by excitation), acetic acid reliable and easy to apply in the field because and anisole (by inhibition) (Kellogg, 1970), only light microscopy is required. Thus, it is and lactic acid (Davis and Sokolove, 1976). very useful for entomologists who conduct The fine structure of sensilla coeloconica epidemiological and vector incrimination (large and small) of An. dirus and An. studies. Further study is needed to deter- cracens appears to be similar in other mine the usefulness of antennal sensilla in mosquito species (Ismail, 1962; Boo and distinguishing all member of the Dirus Mclver, 1976; Pitts and Zwiebel, 2006; Complex. Hempolchom et al., 2017). Using light micro- scopy, the mean number of large sensilla Author Contributions coeloconica on each flagellum of Thai An. Conceptualization, AS; Methodology, KT and dirus (25.33) and Hainan An. dirus (24.78) AS; Resources, PS and PS; Formal Analysis, is significantly greater than in An. cracens KT, RP and AS; Investigation, KT, KA, TY, WJ (19.58). However, the mean number of these and KP; Writing-Original Draft Preparation, sensilla on the antennae of An. dirus and KT and AS; Supervision, WS and AS; Usage of An. cracens is less than the mean number on morphological terminology, REH; Writing- the antennae of An. harrisoni (31.98), An. Review & Editing, REH, PS and AS; Funding minimus (26.25), An. nigerrimus (33.10), Acquisition, KT and AS. An. nitidus (28.73), An. paraliae (37.55), An. pursati (27.85), An. quadriannulatus Funding (29.00) and An. sinensis (36.47) (Gerberg The Chiang Mai University (CMU), Chiang et al., 1994; Wijit et al., 2016; Taai et al., 2017). Mai, Thailand funded the Post-Doctoral Although comparison of the ultrastructure Fellowship 2016 awarded to KT (contract of this sensillum type in the two species number 09/2016). AS was supported by the revealed similar structure, it is important Anandamahidol Foundation (Year 2016– to note that the difference in the number of 2019), the Diamond Research Grant (grant the large sensilla coeloconica present on number PAR-2560-04663), Faculty of their antennae can be used to distinguish Medicine, CMU, and Chiang Mai University them. However, it must be pointed out that through the Center of Insect Vector Study. this difference may not distinguish these species from other members of the Dirus Conflicts of Interest Complex. A comparative study of antennal The authors declare no conflict of interest. sensilla in all members of the complex is The funders had no role in the design of needed to answer this question. the study; in the collection, analyses, or interpretation of data; nor in the writing of the manuscript and the decision to publish CONCLUSIONS the results.

We identified and described, for the first time, Acknowledgments. We would like to thank the fine structure of five types of sensilla on Dr Yukiko Higa for providing the larvae of the the antennal flagella of females of An. dirus An. dirus used to establish the Hainan-strain (Thailand and Hainan strains) and An. colony.

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